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Chimeric protein as well as preparation method and application thereof

A chimeric protein, protein technology, applied in chemical instruments and methods, single-component protein rayon, hybrid peptides, etc., can solve the problem of less fiber, chemical environment, rheological properties and physical pressure of natural spider silk spinning system Insufficient understanding, inability to use practical purposes, etc.

Active Publication Date: 2020-07-28
CHANGCHUN INST OF APPLIED CHEMISTRY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

2) The current spinning process is mostly carried out in an organic solvent environment, which is far from the physiological conditions; and the understanding of the natural spider silk spinning system, including the chemical environment, rheological properties and physical pressure in the body, is insufficient, and it is currently impossible to realize in vitro simulation
In short, currently produced recombinant spider silk fibers produce little or no fibers for practical use due to poor mechanical properties, low reproducibility, or both

Method used

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  • Chimeric protein as well as preparation method and application thereof
  • Chimeric protein as well as preparation method and application thereof
  • Chimeric protein as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Embodiment 1: expression vector construction

[0036] Construction of the coding sequence of the SRT-ELP protein unit (SRT protein coding sequence SEQ ID NO:3+ELP protein coding sequence SEQ ID NO:4), and XbaI restriction site (TCTAGA), homology arm 1 (CTGCAGGAATTCGTTATC, SEQ IDNO :5), NdeI restriction site (CATATG), homology arm 2 (SEQ ID NO: 3 sequence 1-21bp), EcoRV restriction site (GATATC), histidine tag sequence ((CAC) 6 ), the DNA coding sequence corresponding to the stop codon (TAATGA) and the EcoRI restriction site (GAATTC), all elements are connected to obtain gene element 1, XbaI+homology arm 1+NdeI+SRT-ELP protein unit coding sequence+homology arm 2+EcoRV+(CAC) 6 +TAATGA+EcoRI; connect gene element 1 and m13 plasmid by XbaI and EcoRI double digestion to obtain chimeric protein monomer vector (m13-monomer);

[0037] The gene element 2 was obtained from the m13-monomer by XbaI and EcoRV double digestion, and the gene element 2 and the m13-monomer digested by...

Embodiment 2

[0040] Example 2: Protein expression and purification

[0041] The expression vector "pET25b-polymer" was transformed into Escherichia coli competent cells E.coli BLR(DE3). Pick positive clones and culture them in 10ml LB medium (100μg / ml ampicillin) for 8-12 hours (37°C, 220rpm). The volume of the shaker flask is 100ml. 500ml LB medium (200μg / ml ampicillin) in a large shaker flask (volume 500ml) and cultured for 1.5-3 hours (37°C, 220rpm), when the OD600 of the bacterial solution reached 0.5-0.7, add the inducer IPTG (isopropylβ-D -1-thiogalactopyranoside, isopropylthiogalactopyranoside) to a final concentration of 1mM, to induce a large amount of protein expression, and after 4 hours of fermentation (37°C, 220rpm), the bacterial liquid was centrifuged (7000g, 20min, 4°C), and the Bacteria were stored at -80°C.

[0042] The cells were ultrasonically disrupted and centrifuged (11000g, 30min, 4°C) to take the supernatant, which was then passed through a nickel column, a desal...

Embodiment 3

[0043] Embodiment 3: the preparation of biological protein fiber

[0044] Add an aqueous glutaraldehyde solution with an initial mass concentration of 0.1% to an aqueous protein solution (200 mg / ml) of the SRT-ELP polymer, dilute the final concentration of glutaraldehyde to a mass concentration of 0.01%, and crosslink for 10 minutes.

[0045] Squeeze the pre-crosslinked protein aqueous solution into a 0.5% glutaraldehyde aqueous solution (coagulation bath) with a syringe, and use a syringe pump to adjust the advancing speed to 10 μl / min, and use a rotary drum collector at a linear speed of 0.6m. / min speed to collect fibers, the schematic will be figure 2 .

[0046] Dry the collected fibers, soak them in ultrapure water for three seconds until the fibers curl up and become soft, and then take out the fibers and immediately stretch them to 100% of their original length.

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Abstract

The invention relates to the technical field of biological protein fibers, and discloses a chimeric protein as well as a preparation method and application thereof. The chimeric protein provided by the invention is composed of one or more than two ELP-SRT protein units or SRT-ELP protein units. The chimeric protein with a non-natural fiber source and a non-cobweb sequence is designed by utilizinga gene recombination technology from the perspective of bionics, and light and super-strong biological fibers are prepared by crosslinking glutaraldehyde, has the performance exceeding that of many recombinant cobwebs and recombinant silks, and even can be comparable with natural silks and even part of natural cobwebs.

Description

technical field [0001] The invention relates to the technical field of biological protein fibers, in particular to a chimeric protein and its preparation method and application. Background technique [0002] Bio-protein fiber is a new type of fiber formed from natural protein or its structural analogues. It has good biocompatibility, and the production process can achieve sustainable development without the limitation of petroleum resources. Among them, fiber materials derived from biomechanical structural proteins have the advantages of low density, high mechanical strength and strong ductility. Compared with chemical synthetic fibers, its mechanical properties achieve a combination of high strength and high toughness, and it is expected to replace traditional fibers as the next generation of high-tech materials. [0003] At present, the research on high-strength natural protein fibers and their bionics mainly focuses on spider silk, especially dragged silk. Natural spide...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/70D01F4/00C08J3/24C08L89/00C08K5/07
CPCC07K14/43504C12N15/70D01F4/00C08J3/24C07K2319/00C08J2389/00C08K5/07
Inventor 刘凯李敬敬李远鑫张洪杰
Owner CHANGCHUN INST OF APPLIED CHEMISTRY - CHINESE ACAD OF SCI
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